The myofilament subproteome contains both structural and contractile proteins. The thick and thin filament comprise the contractile elements. The thick filament is composed primarily of myosin heavy chain and associated light chains, while the thin filament is composed primarily of actin, tropomyosin (Tm), and the troponin complex (Tn). The Tn complex consists of troponin I (TnI), termed the inhibitory protein due to its ability to block actin-myosin interactions, troponin T (TnT), named for its extensive binding to Tm, and troponin C (TnC) which binds Ca2+ and triggers contraction. The binding of Ca2+ to the low affinity Ca2+-binding sites on TnC results in numerous structural changes that allow the cyclic attachment and detachment of myosin to actin and the subsequent production of force at the expense of ATP hydrolysis (for review see Gordon et al. 2000, Physiol. Rev. 80:853–924 and Tobacman, L. S. 1996, Annu. Rev. Physiol. 58:447–481).
Alterations to the myofilament protein including, but not limited to, de novo expression, up-regulation of selected proteins, down-regulation of selection proteins, mutations, isoform changes and post-translational modifications have been implicated in a variety of diseases, disorders and/or states of injury.
For example, in cardiac muscle specific and selective modification of cardiac troponin I has been proposed as a molecular mechanism that underlies the contractile dysfunction observed in stunning, and other mild reversible forms of ischemia/reperfusion injury (Bolli et al. 1999, Phys. Reviews 79:609–634; McDonough et al. 1999, Circ. Res. 84:9–20; Van Eyk et al. 1998, Circ. Res. 82:261–271; Foster et al. 1999, Circ. Res. 85:470–472; Solaro et al. 1999, Circ. Res. 84:122–124). Protein kinase A, C and G have been shown to collectively phosphorylate cardiac troponin I at five different sites (for review see Biochemistry (Mosc) 1999 64(9):969–85). Further, detection and quantification of cardiac troponin I in serum of patients suffering from acute coronary syndromes has become the most specific and sensitive biochemical marker available for myocardial injury, due to release of the cardiac-specific isoform. Low levels of this protein have also been observed in stable and unstable angina and heart failure.
Mutations in genes encoding skeletal myofilament proteins have been associated with nemaline myopathy, an inherited disorder causing nonprogressive muscle weakness. To date, all identified mutations causing nemaline myopathy and familial hypertrophic cardiomyopathy, an inherited disorder affecting skeletal muscle and heart, respectively, occur within the structural and contractile proteins, as opposed to membrane cytosolic and mitochondrial proteins (Ilkovski et al. 2001 Am. J. Hum. Genet. 68:1333–1343; Michele et al. 2000 J. Mol. Med. 78:543–553; Shaw et al. 2000 Lancet 360(9334):654–5; Bonne et al. 1998 Circ. Res. 83(6):580–93; Marian, A. J. 2002 Curr. Opin. Cardiol. 17(3):242–52).
In diseases such as chronic obstructive pulmonary disease and congestive heart failure, fiber type switching occurs in skeletal muscle. The nature of the switch is dependent upon the muscle group, namely limb versus respiratory. For example, in chronic obstructive pulmonary disease, the diaphragm, which is in continuous use, has an increased proportion of slow oxidative fatigue-resistant fibers (for review see Gayan-Ramirez and Decramer 1996, Rev. Mal. Respir. 17:574–584; Levine et al. 2001, Exerc. Sport Sci. Rev. 29:71–75; Stassijns et al. 1996 Eur. Respir. J. 9:2161–2187), whereas limb muscles have an increased proportion of fast glycolytic fibers (for review see Aliverti and Macklem 2001 Respiration 68:229–239; American Thoracic Society and European Thoracic Society 1991 Am. J. Respir. Crit. Care Med. 159:S1–S40; Mador and Bozkanat 2001 Respir. Res. 2:216–224; Maltais et al. 2000 Clin. Chest Med. 21:5665–677). Fiber-type switching can also be induced during training, for example, in athletes.
Proteolysis of skeletal troponin I has been reported to occur in the diaphragms of severely hypoxemic dogs (Simpson et al. 2000, J. Appl. Physiol. 88:753–760). Further, proteolytic fragments of fast and slow skeletal troponin I were detected in serum samples of a patient with rhabdomyolysis (Simpson et al. 2002, Clin. Chem. 48:1112–1114). These results are suggestive of skeletal myofilament proteins being susceptible to post-translational modifications.
The detection of intact myofilament proteins, degradation products of myofilament proteins, and protein-protein complexes of myofilament proteins, in various biological samples such as blood, tissue, and urine, was described in detail in U.S. patent application Ser. No. 09/115,589, filed Jul. 15, 1998.
Methods for detection of chemical adducts of myofilament proteins (e.g., post-translational modifications) and various modifications thereof, including protein-protein complexes and protein fragments thereof, were described in detail in U.S. patent application Ser. No. 09/419,901, filed Oct. 18, 1999.
The present invention relates to the identification of post-translationally modified myofilament proteins, and in particular phosphorylated troponin I proteins. The phosphorylation state of troponin I proteins is associated with altered contractile function.